First Report of Fusarium concentricum Causing Leaf Blight, Shoot Blight, Flower and Fruit Rot on Kadsura coccinea in China.

Abstract

Kadsura coccinea (Lem.) A. C. Smith is a traditional medicinal plant grown in south China. Lignans and terpenoids extracted from the root, fruit and stem were found to have anti-proliferative, anti-HIV, anti-hepatitis, anti-oxidant, neuroprotective, and other pharmacological attributes for treating rheumatoid arthritis and gastroenteric et al. disorders (Yang et al., 2020). However, little is known about the biotic disorders of this evergreen climbing shrub. In surveys carried out in a 15- hectare -orchard located 23°59'55''N, 113°55'13''E, all K. coccinea plants were observed exhibiting an array of symptoms including leaf blight, shoot blight, flower rot and fruit rot from May to July of 2023, with disease incidences of 17% , 18%, 16% and 28%, respectively. A greyish -brown blight appeared predominantly on the leaf margins or tips. Light to dark brown lesions on the shoots, flower calyxes and fruits were slightly sunken, irregularly shaped, and watery, usually with white aerial mycelium. For pathogen isolation, infected tissues were cut into fragments of about 5 mm in diameter, surface sterilized with 75% ethanol for 15 s and 1% NaClO for 2 min, and then rinsed three times with sterilized distilled water. Filter paper-dried tissues were placed onto potato dextrose agar (PDA) and incubated at 28°C for 5 days. Seventy-one morphologically similar colonies were produced from 100 tissue fragments, with other tissues lacking colonies or with different colonies. Genomic DNA of 10 randomly selected isolates were extracted from 5-day-old pure cultures. Molecular characterization of the 10 subcultured strains was analyzed by sequencing three regions i.e. translation elongation factor 1-alpha (TEF-1), beta-tublin (TUB), and RNA polymerase Ⅱ second largest subunit (RPB2), amplified using primers EF1/EF2 (O'Donnell et al., 1998), T1/T22 (O'Donnell and Cigelnik 1997), and RPB2-5f2/RPB2-7cr (Liu et al. 1999). Maximum likelihood and Bayesian Inference analysis based on the dataset of the combined three sequences by MEGA 11.0 showed that nine strains were consistently identified as F. concentricum. The three sequences (OR632200, OR632199, and OR754282) of a representative strain (SGXF1) shared 99.42%, 99.62%, and 99.80% identity with those of the type strain CBS450. 97. The colonies were white to pale buff with serrated edges and sparse aerial mycelium that initially formed a loose, white cottony texture of 5-10 mm in height. After 7 d's culture at 26°C in an incubator with a photic door, dense and abundant fluffy reddish-white aerial mycelium covered the entire PDA medium of 9 cm-diameter, with alternating pale orange and reddish-grey concentric rings at center with diffusible pigments. Catenate microconidia were obovoid to fusoid-shaped, mostly 0-septate, with a flattened base, (10.98 ± 0.83) x (3.41 ± 0.15) μm (n=20), and were produced on both mono-and polyphialides, whereas macroconidia were curved and long, with a slightly beaked apical cell and a basal cell, mainly 3- to 5-septate, and (33.80 ± 0.81) x (4.31 ± 0.12) μm (n=20) in size. These morphological characteristics further indicates it to be F. concentritum (Nirenberg and O'Donnell, 1998; Xiao et al., 2023). To determine pathogenicity, three 1-y-old seedlings were wound inoculated by conidial suspension (1 × 106 conidia/mL) on different tissues and incubated at 25°C in a greenhouse with 10 replications. At 2 d after inoculation (DAI), the treated flowers gradually turned from fresh red and green to black at calyxes, with the black area then gradually expanding. At 6 DAI, part of the flowers turned black, accompanied by white mycelium and yellowish powdery spots. Similarly, leaves, stems, and fruits turned from green to dark brown at infection sites within 7 DAI. Black to brown, uneven necrotic areas, and reddish spots were observed under a dissecting microscope, with white mycelium attached at 6 DAI. The symptoms observed in lab were similar to those observed in the field, whereas three control plants remained asymptomatic. Fungal colonies identical to initial isolations were recovered from artificially infected tissues and confirmed to be F. concentricum again, thereby completing Koch's postulates. This report is the first to document F. concentricum causing disease on K. coccinea. Appropriate measures will be developed based on this study to protect this economically important plant in the field.